Analysis of the role of IL-21 in development of murine B cell progenitors in the bone marrow

Abstract

IL-21 plays a key role in the late stage of B cell development, where it has been shown to induce growth and differentiation of mature B cells into Ig-secreting plasma cells. Because IL-21R has also been reported on bone marrow (BM) B cell progenitors, we investigated whether IL-21R influenced earlier stages of B cell development. IL-21R is functional as early as the pro-B cell stage, and the strength of receptor-mediated signaling increases as cells mature. The addition of IL-21 to B cell progenitors in cell culture resulted in the accelerated appearance of mature B cell markers and was associated with the induction of Aid, Blimp1, and germline transcripts. We also found that stimulation of both IL-21R and CD40 was sufficient to induce the maturation of early B cell progenitors into IgM- and IgG-secreting cells. Consistent with a role for IL-21 in promoting B cell differentiation, the number of B220(+)CD43(+)IgM(-) pro-B cells was increased, and the number of mature IgM(hi)IgD(hi) cells was decreased in BM of IL-21R-deficient mice. We also report in this paper that IL-21 is expressed by BM CD4(+) T cells. These results provide evidence that IL-21R is functional in B cell progenitors and indicate that IL-21 regulates B cell development.

FIGURE 1

IL-21 is produced by CD4⁺ T cells in BM. A, RT-PCR was performed on cDNA obtained from BM cells of WT, Rag^−/− and TCRβ^−/− mice. B, Freshly isolated BM or BM B220⁻ cells from WT mice were stained with anti-B220, -CD3, -CD4, -CD8, and -NK 1.1 Abs, and sorted into B cells (B220⁺), CD4⁺ T cells (B220⁻CD3⁺CD4⁺NK1.1⁻), CD8⁺ T cells (B220⁻CD3⁺CD8⁺NK1.1⁻), and NK cells (B220⁻NK1.1⁺). RNA was extracted and RT-PCR was performed using primers specific for Il21 and Actin as described in “material and methods”. Spleen was used as a positive control. C, Freshly isolated BM cells from WT mice were stained with anti-CD3, -CD4, -CD44, and –CD69, and sorted into naïve T cells (CD3⁺CD4⁺CD44^loCD69⁻) and memory T cells (CD3⁺CD4⁺CD44^hiCD69⁻) populations. Il21 mRNA expression was measured by Real-time PCR and normalized to β-Actin. Spleen was used as a positive control. The figure is a representative experiment of two independent experiments. D, BM CD4⁺ cells were sorted and cultured with anti-CD3 (10 μg/mL), anti-CD28 (2 μg/mL) with IL-6 (100ng/mL) (solid line) and without IL-6 (100 ng/mL) (dashed line). After 72 hours, IL-21 protein was detected in the supernatant by FACS with beads array. Data presented is a representative experiment that was performed two times with similar results.

FIGURE 2

IL-21R expression on BM B cell progenitors. A, Day 4^IL-7 B220⁺ BM cells were stained with anti-CD2, -κ, and -λ Abs, and sorted into proB (CD2⁻LC⁻), preB (CD2⁺LC⁻), and immature/mature (CD2⁺LC⁺) B cell populations. RNA was extracted and semi-quantitative RT-PCR was performed on serial dilutions of cDNA (undiluted, 1/5 dilution, 1/10 dilution) using primers specific for IL-21R and actin as described in the “materials and methods”. The stromal cell line S17 was used as negative control, and spleen (Sp) cells were used as positive control. Semi-quantitative RT-PCR was performed on four independent sample sets with similar results. B, Surface expression of IL-21R on B cell progenitors. Top panel – Day 4^IL-7 B220⁺ BM cells from WT mice and IL-21R^−/− mice were stained with anti-B220, -CD2, -kappa LC, -lambda LC, and –IL-21Rα. Bottom panel - Freshly isolated BM cells from WT mice and IL-21R^−/− mice were stained with anti-CD2, -κ, -λ, and -IL-21Rα Abs and analyzed by FACS. Open and closed histograms represent binding of anti-mouse IL-21Rα Ab in WT and IL-21R^−/− mice, respectively. Data are representative of two independent experiments.

FIGURE 3

IL-21R stimulation increases tyrosine phosphorylation of STAT1, STAT3 and STAT5 in proB, preB and immature/mature B cells. Day 4^IL-7 B220⁺ BM cells from WT mice were sorted for proB (CD2⁻LC⁻), preB (CD2⁺LC⁻) and immature/mature B (CD2⁺LC⁺) cells. A, Tyrosine-phosphorylated forms of STAT1 (pY701), STAT3 (pY705), and STAT5 (pY694) were detected by Western blotting. Actin represents loading control. B, The IL-21 non-responsive B62c proB cell line was spiked with different numbers of sorted preB cells, stimulated for 15 min. with IL-21 (50 ng/ml), and analyzed for STAT3 phosphorylation by Western blotting. C, B220⁺ BM cells were isolated from RAG^−/− mice and grown in IL-7. On day 4, cells were harvested and stimulated for 15 min with IL-21 (50 ng/ml). Phosphorylation of STAT3 was detected by Western blotting. D, Day 4^IL-7 BM B220⁺ cells from WT and IL-21R^−/− mice were sorted into proB (CD2⁻LC⁻), preB (CD2⁺LC⁻) and immature/mature B (CD2⁺LC⁺) populations. Phosphorylation of STAT3 was detected by Western blotting. E, Day 4^IL-7 BM B220⁺ cells from WT and IL-21R^−/− mice were simulated with IL-7 or IL-21. Phospho-STAT3 was detected by Western blotting. Data are representative of two independent experiments.

FIGURE 4

IL-21 regulates maturation of different B cell progenitors. Day 4^IL-7 B220⁺ BM cells from WT and IL-21R^−/− mice were sorted into proB (CD2⁻LC⁻), preB (CD2⁺LC⁻) and immature/mature B CD2⁺LC⁺) cell populations. Each population was plated with or without IL-21 (30 ng/ml) for 48 hours. Control and IL-21-containing cell cultures initiated with proB (A and B) and preB (C and D) cells from IL-21R^−/− and WT mice were harvested on day 2 of culture and analyzed by FACS using CD2-LC and IgM-IgD Ab combination. AC), Percentages of cells in each population are shown on the plots of a representative experiment. BD), Scatter plot of cell population percentages from separate experiments. Lines connect pairs of experiments performed. Statistical analysis of percentage of preB and immature/mature B cells obtained in control and IL-21-treated WT cell cultures was assessed by 2-tailed paired Student t-tests. The analysis shows that there were a significant increase percentage of preB cells in IL-21 containing culture started with proB cells compare to control (p < 0.0001*) (B; lower panel), and a trend toward an increase percentage of immature/mature B cells in IL-21-containing culture initiated with preB cells (percentage of CD2⁺LC⁺, p=0.09 (D; lower left panel); percentage of CD2⁺LC^hi, p=0.08) (D; lower right panel).

FIGURE 5

IL-21R^−/− mice have more proB and fewer mature B cells than WT mice in BM. BM cells were isolated from WT (n=20) and IL-21R^−/− (n=20) mice and stained for B220, CD43, and IgM; or CD19, CD2, and IgM; or CD19, IgM, and IgD. Percentages (A) and absolute numbers (B) are shown for each cell population. Statistical significance was assessed by 2-tailed Student t test and the level of significance was established at p < 0.05.

FIGURE 6

IL-21 regulates gene expression of Blimp1 and Aid in B cell progenitors. B220⁺ BM cells were grown in IL-7. On day 3, IL-21 (30 ng/mL) was added for 24 hours to half of the culture. Cells were sorted on day 4 into proB (CD2⁻LC⁻), preB (CD2⁺LC⁻) and immature/mature B (CD2⁺LC⁺) cells and total RNA was extracted. Real-time PCR analysis using Blimp1 (A) and Aid (B) specific-primers were performed on cDNA as described in “materials and methods”. Figures show normalized values from a single experiment (left panel) and fold change over untreated from two independent experiments (right panel).

FIGURE 7

IL-21R stimulation induces the expression of γ2b germline transcripts. A, Day 4^IL-7 BM cells were sorted into proB (B220⁺CD2⁻LC⁻), preB (B220⁺CD2⁺LC⁻), and immature/mature B (B220⁺CD2⁺LC⁺) cells. Each population was plated with IL-21 (30ng/mL), with anti-CD40 (2ug/ml) or anti-CD40 and IL21. After 24 hours, total RNA was extracted and RT-PCR analysis was performed to detect GLTγ2b. β-Actin was used to normalize for cDNA input. Band intensity was measured and normalized to β-Actin using Quantity One software. The figure shows one representative experiment that has been performed at least three times with similar results. In pre-B cells stimulated with IL21, the fold increase in GLT levels ranged from 1.12 to 3.11 fold; in immature/mature B cells, the fold increase ranged from 1.23 to 6.25. Statistical analysis of quantitated and normalized band intensity for pre B cells was performed using the Wilcoxian test demonstrating that there was a significant fold increase with IL21 treatment in preB (CD2⁺LC⁻) sorted cells (p=0.032, n=6). B, IL-21 in combination with anti-CD40 induces the formation of Ig secreting cells from cultures initiated from B cells progenitors. Left panel, Day 4^IL-7 B220⁺ BM cells were plated with IL-7 in combination with anti-CD40, IL-21 or both. Supernatants were harvested on day 7 and ELISA was performed as described in “materials and methods”. Right panel, Day 4^IL-7 B220⁺ BM cells were sorted into proB cells (CD2⁻LC⁻) and plated with IL-7 and anti-CD40 with or without IL-21. Culture supernatants were collected on day 9 and ELISA was performed as described in “materials and methods”. Results are representative of three independent experiments.